Posted by
Janne Hyötylä on
Mar 24, 2010; 10:07am
URL: http://imagej.273.s1.nabble.com/Normalizing-background-and-fluorescence-intensity-tp3688820p3688822.html
Dear Dawn,
I second what Mario said. But it might be difficult to find an internal
biological standard for the staining with Alexa488. Is there a possibility
to stain something in the cytoplasm which has always the same
accessibility to Alexa488-antibodies (or are you using some other method
than antibodies?), which does not change during the cell cycle and which
doesn't interfere with your real signal?
You could possibly use the S-phase nuclei as the intensity standard if the
following are true:
* You have at least one S-phase nucleus in each image
* You are sure that the S-phase nuclei should always have the same
intrinsic brightness. I.e. the epitopes have always the same
accessibility, the number of epitopes is always the same, etc.
* Any brightness change is the same for the whole image, i.e. this would
be the case if the mounting procedure has an effect on the brightness or
if the dye itself differs from batch to batch. If for example the
brightness of the dye somehow depends on the local environment, then this
is not the case.
From the above points actually follows that all S-phase nuclei in one
image should have exactly the same brightness in image after background
subtraction.
As you can see, to do something quantitative is indeed quite tricky and
you have to consider all pitfalls carefully, and be aware of the
assumptions and implications when using any quantitative method.
As for how to do it, once you have your internal brightness standard (e.g.
the S-phase nuclei), you should do a background subtraction as described
by Mario (again, many possible ways with their own implications). Then
measure the intensity of your standard (normally using the ROI manager and
"Measure..." is good, depends how your images look like) and divide the
image by this value (Process > Math...). You can convert your image to
32-bit before normalizing to avoid loss of information.
Best,
Janne
On Wed, 24 Mar 2010 06:34:22 +0100, Mario Faretta
<
[hidden email]> wrote:
> Dear Dawn,
> there are a lot of factors to be considered in your problem. Variations
> in
> intensity can be related to the way you acquire images (depending on the
> setting of your microscope, filters, lamp alignment...) or to the
> preparation
> of the samples (efficiency of the staining, batch of reagents,...).
> It could be a tricky stuff to compare "quantitatively" different
> coverslips
> and preparations.
> To correct the first factors you can try to apply a "flatfield"
> correction to
> make illumination homogeneous (in the plugin section on the ImageJ
> website you
> can find some solutions with good explanations and you can also search
> archive
> of the list for the topic). You can use empty fields of view (no cells)
> to
> acquire images for background subtraction using the image calculator or
> to
> apply one of the background functions.
> For the second story, i.e. normalize signals for comparative
> measurements, the
> best thing is probably to have an internal "biological" standard to
> refer as
> unit of measure of your experiments...
> Hope it helps
> Mario
>
> Dawn Koh (
[hidden email]) wrote:
> >
> > Hi all,
> >
> > Im currently working on labeling DNA with Alexa Fluor Dye 488. In
> my images
> > I can see nuclei
> > with maximum fluorescence at S-phase and slight fluorescence when
> undergoing
> > DNA repair.
> > This is actually another method to assay for unscheduled DNA
> synthesis (UDS)
> > instead of using radioisotopes.
> >
> > My problem begins when I try to compare images from different
> views or cover
> > slips.
> > As I try to compare nuclei at S phase from different cover slips,
> > I notice that the fluorescence intensity slightly stronger for one
> cover
> > slip and weaker for the other.
> > I need both cover slips to have the same intensity in order to
> compare the
> > nuclei undergoing DNA repair.
> > Does anyone know how I can normalize the images on Image J so that
> they can
> > be compared fairly?
> >
> > Thanks!
> >
> > Best Regards
> > Dawn
> >